Tremendous success fighting HIV infection with highly active antiretroviral therapy turns AIDS into a manageable clinical entity. However, nucleoside reverse transcriptase inhibitors (NRTIs), the indispensable component of the treatment, have displayed severe adverse reaction mostly manifested as mitochondrial toxicities. The toxicities have been implicated to NRTIs inhibitory effect on human mitochondrial DNA polymerase gamma (DNA Pol 3). We propose to elucidate structural basis of NRTIs mitochondrial toxicity by conducting structural studies of human Pol 3 captured at various stages of DNA replication. Specifically, we will determine crystal structures of Pol 3 during replication and structures of a stalled replicating Pol 3 by NRTIs zalcitabine and AZT. Comparison of these structures will provide molecular basis for NRTIs mitochondrial toxicity. We have recently obtained crystals of human Pol 3 holoenzyme containing the catalytic and accessory subunits that diffracted to 3.1 E resolution. In addition, we have obtained crystals of Pol 3 complexed with a primer-template DNA duplex, as well as a complex of Pol 3-DNA with zalcitabine. Upon completion of the proposed studies, we will provide an array of atomic-resolution snapshots of Pol 3 caught in action of DNA replication or inhibition. Comparison of inhibitor interactions with human Pol 3 with that of HIV reverse transcriptase will provide invaluable guidance in design high potency and low toxic antiviral reagents. PUBLIC HELATH RELEVANCE: Tremendous success fighting HIV infection with highly active antiretroviral therapy turns AIDS into a manageable clinical entity. However, nucleoside reverse transcriptase inhibitors (NRTIs), the indispensable component of the treatment, have displayed severe adverse reaction mostly manifested as mitochondrial toxicities. As AIDS epidemic continues and significantly increased patients survival duration, the side effects due to long-term usage of NRTIs become increasingly more common. Studies have implicated the NRTIs adverse reaction to inhibition of human mitochondrial DNA polymerase that replicates and repairs mitochondrial DNA. Active DNA replication is critical to the integrity of the organelle. Interference with human Pol 3 activity causes mutations and DNA depletion, resulting myopathy, cardiomyopathy, lactose acidosis and encephalopathy. We propose to conduct structural studies of NRTIs inhibitory mechanism of human mitochondrial DNA polymerase. The results of the study will not only increase our understanding of mitochondrial DNA replication, but also will be invaluable to aid design of high potency and low toxic anti- HIV reagents.